The Notch receptor family is a class of evolutionarily conserved transmembrane receptors that transmit signals affecting development in organisms as diverse as sea urchins and humans. Notch receptors and their ligands Delta and Serrate (known as Jagged in mammals) are transmembrane proteins with large extracellular domains that contain epidermal growth factor (EGF)-like repeats. The number of Notch paralogues differs between species. For example, there are four Notch receptors in mammals (Notch1-Notch4), two in Caenorhabditis elegans (LIN-12 and GLP-1) and one in Drosophila melanogaster (Notch). Notch receptors are proteolytically processed during transport to the cell surface by a furin-like protease at a site S1, which is N-terminal to the transmembrane domain, producing an extracellular Notch (ECN) subunit and a Notch transmembrane subunit (NTM). These two subunits remain non-covalently associated and constitute the mature heterodimeric cell-surface receptor.
Notch2 ECN subunits contain 36 N-terminal EGF-like repeats followed by three tandemly repeated Lin 12/Notch Repeat (LNR) modules that precede the S1 site. Each LNR module contains three disulfide bonds and a group of conserved acidic and polar residues predicted to coordinate a calcium ion. Within the EGF repeat region lie binding sites for the activating ligands.
The Notch2 NTM comprises an extracellular region (which harbors the S2 cleavage site), a transmembrane segment (which harbors the S3 cleavage site), and a large intracellular portion that includes a RAM23 domain, six ankyrin repeats, a transactivation domain and a carboxy-terminal PEST sequence. Stable association of the ECN and NTM subunits is dependent on a heterodimerization domain (HD) comprising the carboxy-terminal end of the ECN (termed HD-N) and the extracellular amino-terminal end of NTM (termed HD-C). Before ligand-induced activation, Notch is maintained in a resting conformation by a negative regulatory region (NRR), which comprises the three LNRs and the HD domain. The crystal structure of the Notch2 NRR is reported in Gordon et al., (2007) Nature Structural & Molecular Biology 14:295-300, 2007.
Binding of a Notch ligand to the ECN subunit initiates two successive proteolytic cleavages that occur through regulated intramembrane proteolysis. The first cleavage by a metalloprotease (ADAM17) at site S2 renders the Notch transmembrane subunit susceptible to a second cleavage at site S3 close to the inner leaflet of the plasma membrane. Site S3 cleavage, which is catalyzed by a multiprotein complex containing presenilin and nicastrin and promoting γ-secretase activity, liberates the intracellular portion of the Notch transmembrane subunit, allowing it to translocate to the nucleus and activate transcription of target genes. (For review of the proteolytic cleavage of Notch, see, e.g., Sisodia et al., Nat. Rev. Neurosci. 3:281-290, 2002.)
Five Notch ligands of the Jagged and Delta-like classes have been identified in humans (Jagged1 (also termed Serrate1), Jagged2 (also termed Serrate2), Delta-like1 (also termed DLL1), Delta-like3 (also termed DLL3), and Delta-like4 (also termed DLL4)). Each of the ligands is a single-pass transmembrane protein with a conserved N-terminal Delta, Serrate, LAG-2 (DSL) motif essential for binding Notch. A series of EGF-like modules C-terminal to the DSL motif precede the membrane-spanning segment. Unlike the Notch receptors, the ligands have short cytoplasmic tails of 70-215 amino acids at the C-terminus. In addition, other types of ligands have been reported (e.g., DNER, NB3, and F3/Contactin). (For review of Notch ligands and ligand-mediated Notch activation, see, e.g., D'Souza et al., Oncogene 27:5148-5167, 2008.)
The Notch pathway functions during diverse developmental and physiological processes including those affecting neurogenesis in flies and vertebrates. In general, Notch signaling is involved in lateral inhibition, lineage decisions, and the establishment of boundaries between groups of cells. (See, e.g., Bray, Mol. Cell Biol. 7:678-679, 2006.) A variety of human diseases, including cancers and neurodegenerative disorders have been shown to result from mutations in genes encoding Notch receptors or their ligands. (See, e.g., Nam et al., Curr. Opin. Chem. Biol. 6:501-509, 2002.)
Certain anti-Notch2 antagonist antibodies having therapeutic efficacy have been described. (See U.S. Patent Application Publication No. US 2009/0081238 A1, expressly incorporated by reference in its entirety herein.) For example, such antibodies bind to the negative regulatory region (NRR) of Notch2, block Notch2 signaling, and inhibit the growth of melanoma cell lines, diffuse large B-cell lymphoma (DLBCL) cell lines, and marginal zone B cells. Certain anti-Notch2 antibodies described therein bind to the LNR-A domain (the first of the three LIN12/Notch Repeats) and the HD-C domain of Notch2 NRR.
Adult liver has the capacity to regenerate after injury. It has been speculated that biliary-hepatocyte progenitor cells (oval cells) in or near intrahepatic bile ducts can differentiate into adult hepatocytes (Brues and Marble, J. Exp. Med., 65(1):15 (1937); Zajicek et al., Liver, 5(6):293 (1985)), which subsequently mature as they move toward the central vein and eventually undergo apoptosis and elimination (Benedetti et al., J. Hepatol., 7(3):319 (1988)). Recent lineage-tracing studies have supported a role of progenitor cells in liver homeostasis and repair, but the signals that govern precursor differentiation into hepatocytes are poorly understood. While Notch signaling is known to be critical for the proper formation of the intrahepatic biliary system during development (Lozier et al., PLoS One 3(3):e1851 (2008); McCright et al., Development 129(4):1075 (2002)), it was not known what role, if any, Notch signaling plays in adult hepatocyte formation and in adult hepatobiliary disease.
Chronic liver disease is marked by gradual destruction of liver tissue, especially of hepatocytes and the functional lobular unit, leading to fibrosis (replacement of liver tissue with scar tissue) and cirrhosis (fibrosis with ineffective nodular regeneration and associated loss of liver function). Moreover, chronic liver disease often includes pathological biliary hyperplasia and may increase the risk of liver cancer.
There is a need in the art for further therapeutic methods of treating liver conditions. The invention described herein meets the above-described needs and provides other benefits.